The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury

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Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in humans has been explored over the past decade the data remain controversial in terms of optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study aimed to explore the potential of different rTMS protocols to induce changes in gene expression in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the expression of 96 genes were examined using low-density expression arrays after MCAO alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS) theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2-day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced significant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of iTBS induced significant increase in the expression of 52 genes. There were no downregulated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS effects were negligible. Upregulated genes included those involved in angiogenesis, inflammation, injury response and cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute ischemic-reperfusion brain injury induces complex changes in gene expression that span multiple pathways, which generally promote the recovery. They also demonstrate that induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pattern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of rTMS application in stroke may be to prime the brain, enhancing its potential to cope with the injury and to rewire. This could further augment its potential to favorably respond to rehabilitation, and to restore some of the loss functions.

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Citation: Ljubisavljevic MR, Javid A, Oommen J,
Parekh K, Nagelkerke N, Shehab S, et al. (2015) The
Effects of Different Repetitive Transcranial Magnetic
Stimulation (rTMS) Protocols on Cortical Gene
Expression in a Rat Model of Cerebral IschemicReperfusion Injury. PLoS ONE 10(10): e0139892.
doi:10.1371/journal.pone.0139892

Editor: Johannes Boltze, Fraunhofer Research
Institution of Marine Biotechnology, GERMANY

Received: December 8, 2014

Accepted: September 18, 2015

Published: October 2, 2015

Copyright: © 2015 Ljubisavljevic et al. This is an
open access article distributed under the terms of the
[Creative Commons Attribution License, which permits](http://creativecommons.org/licenses/by/4.0/)
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.

Data Availability Statement: All relevant data are
within the paper.

Funding: This work was supported by a United Arab
Emirates University Grant 31M016 to Milos
Ljubisavljevic.

Competing Interests: The authors have declared
that no competing interests exist.

RESEARCH ARTICLE
# The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury

Milos R. Ljubisavljevic 1 *, Asma Javid 1, Joji Oommen 1, Khatija Parekh 1,
Nico Nagelkerke 2,3, Safa Shehab 4, Thomas E. Adrian 1

1 Department of Physiology, College of Medicine and Health Sciences, UAE University, Al Ain, UAE,
2 Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Queen Elizabeth Central Hospital,
College of Medicine, Blantyre, Malawi, 3 Department of Community Medicine, College of Medicine and
Health Sciences, UAE University, Al Ain, UAE, 4 Department of Anatomy, College of Medicine and Health
Sciences, UAE University, Al Ain, UAE

- milos@uaeu.ac.ae

## Abstract

Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in

humans has been explored over the past decade the data remain controversial in terms of

optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study

aimed to explore the potential of different rTMS protocols to induce changes in gene expres
sion in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by

middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the

expression of 96 genes were examined using low-density expression arrays after MCAO

alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS)

theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2
day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced signifi
cant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of

iTBS induced significant increase in the expression of 52 genes. There were no downregu
lated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS

effects were negligible. Upregulated genes included those involved in angiogenesis,

inflammation, injury response and cellular repair, structural remodeling, neuroprotection,

neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute

ischemic-reperfusion brain injury induces complex changes in gene expression that span

multiple pathways, which generally promote the recovery. They also demonstrate that

induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pat
tern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of

rTMS application in stroke may be to prime the brain, enhancing its potential to cope with

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 1 / 25

rTMS Effects on Gene Expression in Stroke in Rats

the injury and to rewire. This could further augment its potential to favorably respond to

rehabilitation, and to restore some of the loss functions.

Introduction

Transcranial Magnetic Stimulation (TMS) is a well-established, non-invasive technique that
allows the assessment and modulation of brain excitability. Repetitive TMS (rTMS), a variant
of TMS that involves repeated application of TMS pulses, may facilitate or suppress brain activity with variable behavioral effects. Research generally shows that the functional effects of
rTMS on cortical excitability depend on stimulation intensity, frequency and the overall stimulation pattern. It appears that rTMS repeated at fixed high-frequency intervals (> 4 Hz)
increase cortical excitability, while stimuli repeated at low-frequency (~ 1Hz) decrease it [1].
rTMS protocols utilizing patterned stimulation like theta-burst patterns (bursts of 3–5 pulses
at 50–100 Hz, repeated at 5 Hz, i.e. theta rhythm) appear to enhance cortical excitability if
applied continuously (cTBS), whereas if applied intermittently (iTBS) they tend to lower cortical excitability [2]. Furthermore, changes in cortical excitability elicited by rTMS may outlast
the duration of the stimulation [3], a finding that has prompted considerable exploration of the
potential of rTMS neurological and psychiatric therapy. Limited but promising data currently
exist for the benefit of the rTMS in the treatment of depression, tinnitus, anxiety disorders,
neurodegenerative diseases and pain syndromes [4]. In stroke, rTMS was also applied based on
a model of interhemispheric competition for sensory and motor processing [5–7], prompting
development of two conceptually different stimulation strategies [8,9]: one aiming to increase
excitability of the affected hemisphere by excitatory rTMS [10–12] and the other aiming to
suppress excitability of the unaffected hemisphere by inhibitory rTMS [13–16]. In patients
with chronic post-stroke hemiparesis, for example, stimulation of the affected motor cortex
with 5 Hz [17] or 10 Hz [10] facilitated practice-dependent plasticity and improved motor
learning, whereas inhibition of the contra-lesional hemisphere with 1-Hz rTMS also enhanced
motor recovery [13,14,17].

The current understanding of mechanisms aiding functional recovery after stroke suggest
complex mechanisms that involve resolution of edema and necrotic tissue, reperfusion of the
ischemic penumbra [18] and a set of neuronal compensatory mechanisms. These mechanisms
include the recruitment of new/additional pathways, disinhibition of redundant neuronal connections and formation of new neural networks to take over function of the damaged areas

[19]. What remains unclear are the effects of rTMS and how it interacts with such complex cellular and molecular milieu. In animals, rTMS increases the content of ATP and microtubule
associated protein-2 expression [20] while promoting the recovery of the neuronal function

[21], enhancing the long-term potentiation of the hippocampal neurons [22], preventing ischemic neural damage [23], enhancing anti-apoptotic mechanisms in the peri-ischemic area [24]
and inducing neuroprotective effects [25].

However, in human studies, variable effects of rTMS interventions were reported [26,27].
The duration of the effects also seems to vary and depend on several factors, including the timing of the rTMS application (subacute or chronic stroke), the patient’s characteristics and the
site of stimulation [28–30].The possibility of varying rTMS parameters (intensity, pattern,
duration) makes the potential effects and therapeutic outcomes even more unpredictable. Furthermore, the effectiveness of rTMS may be influenced by the nature of the underlying pathological processes. A common assumption is that therapeutic effects in patients can be predicted

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 2 / 25

rTMS Effects on Gene Expression in Stroke in Rats

based on the modulatory effects of rTMS in healthy subjects. However, there is no real evidence
to suggest that this is always the case because the susceptibility to the conditioning effects of
rTMS may well depend on the underlying pathology. Finally, it is apparent that the long-term
clinical improvements caused by rTMS cannot be entirely explained by immediate electrophysiological processes caused by rTMS. Rather, processes beyond instantaneous electrophysiological modulation of neuronal activity related to adaptive changes in gene expression may be
involved in sustaining rTMS effects.

This study primarily aimed to examine whether different rTMS protocols have differential
effects on gene expression in lesioned cortices after ischemic-reperfusion brain injury. Thus,
we examined the effects of four standard rTMS protocols (1Hz, 5 Hz, cTBS and iTBS) on functional recovery and changes in gene expression in lesioned rat cortices with subacute cerebral
ischemic-reperfusion injury induced by middle cerebral artery occlusion (MCAO). We
assessed changes in the expression of 98 genes known to be altered by stroke, as well as those
potentially involved in promoting recovery after stroke, with real-time RT-PCR after two
weeks of rTMS.

Methodology

Ethics statement

All experiments and procedures were carried out according to the National Institute of Health
(NIH) guidelines for the care and use of laboratory animals and approved by the appropriate
local or national ethics board (permit number A06/11 from the Animal Ethics Committee of
the College of Medicine and Health Sciences, UAE University). All animals were singly housed
in cages under standard conditions (12 hour light-day cycle) with free access to water and food
before and after all procedures. All efforts were made to minimize animal suffering and to
reduce the number of animals used.

Animals and experimental groups

A total of 149 male Wistar rats weighing 260–270 g were used for this study. Out of 104 animals that underwent middle cerebral artery occlusion-reperfusion (MCAO), 87 rats survived,
providing a survival rate of 84%. Excluded from the experiment were rats not showing hemiplegia, neurological deficits (at 48 hours, see later behavioral testing) or a clearly visible
infarcted area two weeks after MCAO (n = 6). Consequently, 81 rats were randomly assigned
to the following nine MCAO groups (n = 9 animals/group): four rTMS MCAO groups (1 Hz,
5Hz, cTBS and iTBS stimulation groups), four rTMS MCAO sham-stimulated groups
(ShSTIM) using the same rTMS protocols, and one MCAO group. In addition, 18 animals
were used in control (CON) (n = 9) and sham-operated (ShMCAO) group (n = 9). The
remaining animals (n = 27) were used to assess the stroke volume, as will be discussed later.

MCAO surgery

We used a unilateral middle cerebral artery occlusion (MCAO) model of brain infarction
described in previous studies, with minor modifications. Anesthesia was administrated as an
intramuscular injection, a combination of ketamine (110 mg/kg) and xylazine (10mg/kg)
(Troy Laboratories, NSW, Australia). When necessary, this anesthesia was followed with maintenance doses depending on the reflex withdrawal response and breathing rate. Artificial tears
ointment was applied to the animals’ eyes for protection and lubrication. Using a homoeothermic blanket control unit and feedback-regulated heating lamps, the animals’ temperature was
monitored and maintained between 37 and 37.5°C during and immediately after the surgical

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rTMS Effects on Gene Expression in Stroke in Rats

procedure. A midline neck incision of the skin was performed to reveal the left common, external (ECA) and internal (ICA) carotid artery. A 4–0 monofilament nylon suture with a silicon
coated tip (Doccol Co-operation, Redlands, CA, USA) was introduced through a small incision
in ECA and advanced into the ICA occluding the origin of the middle cerebral artery. After 90
minutes of occlusion, the monofilament was removed and the ECA permanently tied. The animals were then placed in individual cages and carefully monitored until they started drinking
and eating spontaneously. Sham-operated animals were treated similarly except for the
MCAO.

Behavioral testing

Functional deficits after MCAO and rTMS were assessed by rating the severity of deficits with
a series of simple neurological tests combined into a composite Behavior Deficit Score (BDS)

[31]. BDS included walking initiation, spontaneous circling, rotation, righting, tail hang, paw
placement, horizontal bar and postural reflexes. Each test was scored on an ordinal scale, based
on a set of pre-determined criteria described elsewhere [31–33] with total BDS ranging from
0–21, where 0 indicates fully expressed deficit and 21 corresponds to normal animal. In order
to reduce variance and increase data reliability the tests were repeated twice to three times,
depending on the test [34]. Functional deficit was also scored on a modified 0-5-point Bederson scale (BS) [35]. BS expresses the degree of gross neurological impairment on an ordinal
scale, where 0 indicates no deficit, 1 indicates mild forelimb weakness, 2 indicates severe forelimb weakness and consistent rotation to the side of deficit when lifted by tail, 3 indicates spontaneous circling or walking to the contralateral side, 4 indicates walking only when stimulated
or depressed level of consciousness, and 5 signifies animal unresponsive to stimulation. MCAO
animals that received a score of 0 (i.e. no deficit) at 48 hours post-occlusion were excluded
from the study. Behavioral changes were evaluated 24 hours before (-24h) and 48 hours after
MCAO (48h), as well as after the last rTMS session 17 days after MCAO (17d) (see Fig 1). The
examiner performing behavioral testing was blinded for the stimulation (real vs. sham) and
MCAO (real vs. sham-operated).

Stroke assessment

To assess the size of lesioned area and the consistency of the stroke size brains from randomly
selected animals with MCAO (n = 6) were stained 48 hours later using triphenyl tetrazolium
chloride (TTC) stain. In addition, TTC staining was used to confirm the absence of lesion in
sham-operated animals (n = 3). To assess the effects of rTMS on the size of lesioned area additional 14 animals were randomly assigned into MCAO (n = 7) and iTBS (n = 7) group and
their brains stained on day 17.

Fig 1. Timeline of the MCAO-rTMS experiments (BS—5-point neurological deficit Bederson scale, BDS–Behavioral Deficit Score).

doi:10.1371/journal.pone.0139892.g001

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rTMS Effects on Gene Expression in Stroke in Rats

For TTC staining, the animals were guillotined, the brains were dissected out and 2mm coronal slices were cut using a brain matrix (Braintree Scientific, MA; USA). The slices were then
stained with 2% TTC (Sigma Inc.), dissolved in saline for 30 min at 37° C. The slices were then
fixed in 10% formalin overnight. Each section was scanned using a stereomicroscope (Leica
MZ16A, Heerbrugg; Switzerland), and the size of the pale injured area versus deep red color
non-injured area was marked and calculated using the indirect method [36] (Leica Application
Suite v 3.0, Heerbrugg; Switzerland).

To further confirm the character of the MCAO immunohistochemistry for parvalbumin,
calbindin and calretinin was performed on free floating sections (n = 4). Initially, rats were perfused using 4% paraformaldehyde 2 hours after termination of rTMS. Brains were cut coronally
at 70μm thickness using a vibratome. Brains sections were incubated with polyclonal rabbit
anti-parvalbumin (Cat #PV-25; Swant, Switzerland; 1:20,000), polyclonal rabbit anti-calbindin
(Cat# D-28K; Swant, Switzerland; 1:20,000) and polyclonal rabbit anti-calretinin (Cat# 7699/
3H; Swant, Switzerland; 1:10,000) overnight. After rinsing in phosphate buffered saline PBS,
the sections were incubated in biotinylated goat anti-rabbit IgG (1:500) for 1 hour then in
extravidin perioxidase conjugate (Sigma, 1:1000) for another hour. To visualize immunoreactivity the sections were incubated for 5 minutes in a solution of 25 mg diaminobenzidine
(DAB) in 50 ml 0.1 M phosphate buffer (PB, pH 7.4) with 7.5μl hydrogen peroxidase (30%)
and 1 ml nickel chloride (3.5%) added to intensify the reaction. Finally the sections were rinsed
in PB and mounted on gelatin coated slides. Later sections were dehydrated in alcohol, cleared
in xylene and mounted with DPX. All antibodies were diluted in PBS containing 0.3% triton.

rTMS and experimental groups

Four rTMS protocols were used: 1Hz, 5Hz, cTBS and iTBS. During the stimulation, rats
were un-anesthetized, wrapped with their eyes partly covered with a thick cloth and gently
restrained on a platform with Velcro straps. To reduce stress related to handling, restraining,
and stimulation, rats were familiarized to these conditions (including rTMS noise) one week
prior to surgery.

rTMS was started 3 days after MCAO ischemic injury (Fig 1). Rats were stimulated once per
day, between 9 and 11 am, for 10 days (two weeks with a 2-day break) using the standard figure-of-eight coil (MagPro Magnetic Stimulator, Farum, Denmark), positioned and oriented to
induce synaptic activity in most cortical areas of the left hemisphere [37]. Initially, the motor
threshold was grossly estimated by adjusting the stimulation intensity for each animal so that it
barely evoked twitches of hind limbs and body muscles [38]. Thereafter, the stimulation was
increased by 10%. The mean stimulus strength was 30.0±1.5% of maximal stimulator output
strength (range 28–32%). The motor threshold was randomly checked at the beginning of the
stimulation session for each animal. There were no significant changes in thresholds between
stimulated groups during the course of the experiment. The selected stimulation intensity did
not induce any visible discomfort to the animals; they remained generally restful throughout
the experimental sessions.

The animals in each rTMS group received 2400 pulses per day. The number of pulses was
chosen to mimic, as closely as possible, the stimulation rTMS protocols used in human studies.
To exclude the potential effects of duration, the daily stimulation protocol was divided into
four blocks, with 600 pulses/block. The time between the blocks was adjusted to keep the total
duration of stimulation between 50 and 55 min for all protocols. In 1 Hz rTMS protocol four
10 minute blocks, with a four-minute pause in-between were given, while in 5 Hz rTMS protocol a continuous 2 min stimulation repeated at 15 min intervals was given. In iTBS, four blocks
of ten 50 Hz bursts (3 pulses each), repeated 20 times at 5Hz intervals (600 stimuli), were given

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rTMS Effects on Gene Expression in Stroke in Rats

at 15 min intervals. For cTBS, the same burst (3 stimuli at 50 Hz, repeated at 5 Hz intervals)
was given continuously in 20 s train, repeated four times at 15 min intervals. For sham-stimulated groups, we used the same stimulation protocols except that the coil was placed 15 cm
above the rat’s head. For more information on rTMS protocols and their immediate effects on
excitability in normal human brains, see Huag et al. (2005, 2009) and Fitzgerald et al. (2005)

[1,2,39].

Low-density gene expression array

Animals were guillotined and brain tissue was immediately placed in RNAlater (AM7021,
Applied Biosystems, Carlsbad, CA, USA) and kept overnight at 4°C to allow thorough penetration of the tissue. Subsequently, a block of tissue a few millimeters thick was taken after the
brain was coronally transected with a scalpel. The area from which the tissue was taken was
determined according to each animal’s individual distribution of infarction. This area was easily detectable two weeks after MCAO, as the infarcted area had a clearly different color (whitened) from the rest of the tissue. Typically, the samples included the peri-infarct area (outer
borders of the infarction also containing some healthy tissue), and a small portion of the
infarcted area. We also sampled brain tissue from the contralateral hemisphere homologous to
the infarct area and the peri-infarct area. Tissue samples were then frozen at -80°C pending further processing. Isolation of total RNA from the tissue was performed using the SV Total RNA
Isolation System (Promega, Madison, WI; USA) according to the manufacturer’s instructions.
We determined the concentration and purity of the RNA samples by measuring the absorbance
at 260 nm (A260) and the ratio of the absorbance at 260 and 280 nm (ND-1000 NanoDrop).
We performed gene expression analysis using custom pre-loaded Taqman Low Density Arrays
(TLDA) (Format 96a, Applied Biosystems, Foster City, CA; USA) under universal cycling conditions (10 min 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C). We loaded assays on the
TLDA panel with cDNA samples from each individual animal. Each port on the TLDA was
loaded with 50μl of cDNA at a concentration of (4ng/μl) and 50μl of 2X Master Mix. Table 1
shows the target genes surveyed in the plates. We selected the reference gene by examining the
representative samples from each group. The 18S gene showed the highest stability of expression across groups (mean cycle number of 11.85±0.42). We selected a calibrator from the sample with the lowest Ct value, from the control sample for control vs. MCAO comparison and
from the MCAO sample for MCAO vs. rTMS treatment comparison. Data were analyzed
using the comparative CT (2 -ΔΔCt ) method with a relative quantification RQmin/RQmax confidence set at 95% [40].

Quantitative PCR

To confirm changes seen on low density arrays, we analyzed changes in expression of selected
single genes by quantitative PCR (qPCR) using TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA). We performed the reactions according to standard protocols, using
the following gene expression assays: Gria2, BDNF and Gabbr1. We used 18S rRNA as a control, and ran each cDNA sample (n = 4/gene) in triplicate. Control wells with no template were
included on each plate to check for contamination. Data were analyzed as described above
using the comparative CT (2 -ΔΔCt ) method.

Statistics

Data were evaluated for normality. Physiological data were analyzed by mixed factor ANOVA.
The difference in infarct volume between MCAO and iTBS treated animals was analyzed using
two-tailed unpaired student’s t-test. The difference in neurologic deficits on 5-point interval

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rTMS Effects on Gene Expression in Stroke in Rats

Table 1. Fold-changes in mRNAs (RQs) of all genes that showed expression on the microarray in MCAO and ShMCAO group using the control
group as a calibrator. C and Sh in the group significance column indicate significant difference to CON and ShMCAO group, respectively.

Gene
Symbol

Gene name Assay ID MCAO Group
Sig. Diff.

ShMCAO Variance Statistic

(F)

df1 df2 Sig.

Actb Actin beta Rn00667869_m1 1.50 C Sh 0.92 0.011 12.219 2 13.727 0.001

Adcy1 Adenylate cyclase 1 Rn02115682_s1 0.64 C Sh 0.98 0.975 4.651 2 14.583 0.027

Adcy8 Adenylate cyclase 8 (brain) Rn00567592_m1 0.76 0.88 0.002 0.650 2 12.238 0.539

Adm Adrenomedullin Rn00562327_m1 14.74 C Sh 0.95 0.001 18.484 2 12.441 0.0001

Adrb1 Adrenergic beta-1-receptor Rn00824536_s1 0.68 C 1 0.209 5.783 2 13.468 0.015

Ak1 Adenylate kinase 1 Rn00577377_m1 1.1 0.9 0.060 0.736 2 13.699 0.497

Angpt1 Angiopoietin 1 Rn00585552_m1 8.08 C Sh 0.85 0.302 86.168 2 13.892 0.0001

Angpt2 Angiopoietin 2 Rn01756774_m1 26.01 C Sh 0.84 0.003 30.380 2 12.088 0.0001

Apoe Apolipoprotein E Rn00593680_m1 4.5 C Sh 0.93 0.000 22.946 2 12.610 0.0001

App Amyloid beta (A4) precursor

protein

Rn00570673_m1 1.22 0.94 0.213 1.827 2 13.822 0.198

Arrb1 Arrestin beta 1 Rn00563760_m1 1.09 0.93 0.110 0.771 2 13.942 0.481

Arrb2 Arrestin beta 2 Rn00563775_m1 1.58 1.02 0.188 2.290 2 13.452 0.139

Atf3 Activating transcription factor 3 Rn00563784_m1 24 C Sh 1.25 0.007 53.455 2 13.380 0.0001

Bag3 Bcl2-associated athanogene 3 Rn01754954_m1 3.5 C Sh 0.92 0.001 19.470 2 13.281 0.0001

Bai1 Rct59893.0 Rn01504966_m1 0.68 Sh 0.97 0.349 4.276 2 14.350 0.035

Bdnf Brain-derived neurotrophic factor Rn00560868_m1 0.39 C 1.01 0.027 6.287 2 9.858 0.017

Btg2 BTG family member 2 Rn00568504_m1 0.92 0.98 0.368 0.058 2 10.887 0.944

Car11 Carbonic anhydrase 11 Rn00598344_m1 1.03 0.93 0.018 0.351 2 13.847 0.710

Cck Cholecystokinin Rn00563215_m1 1.31 0.96 0.193 1.690 2 13.402 0.222

Cdk5 Cyclin-dependent kinase 5 Rn00590045_m1 1.33 0.92 0.061 2.775 2 12.533 0.101

Cited2 Cbp/p300-interacting transactivator Rn00586705_m1 1.33 C 1 0.546 4.766 2 14.130 0.026

Clock Clock homolog (mouse) Rn00573120_m1 1.08 0.91 0.014 0.858 2 13.731 0.445

Cnr1 Cannabinoid receptor 1 (brain) Rn00562880_m1 1.34 0.91 0.857 1.846 2 14.576 0.193

Creb1 Camp responsive element binding

protein 1

Rn00578826_m1 1.51 C Sh 0.94 0.256 6.939 2 14.287 0.008

Cryab Crystallin alpha B Rn00564026_m1 2.07 C Sh 1.01 0.502 17.617 2 13.981 0.0001

Csnk1e Casein kinase 1 epsilon Rn00581130_m1 1.47 C Sh 0.93 0.122 7.520 2 12.963 0.007

Cxcl12 Chemokine (C-X-C motif) ligand 12 Rn00573260_m1 6.22 C Sh 0.93 0.001 18.006 2 13.010 0.0001

Cxcr4 Chemokine (C-X-C motif) receptor Rn01483207_m1 7.63 C Sh 0.88 0.001 17.944 2 12.105 0.0001

Dagla Diacylglycerol lipase alpha Rn01454304_m1 0.87 0.99 0.623 0.281 2 14.363 0.759

Dbh Dopamine beta-hydroxylase Rn00565819_m1 0.53 C Sh 0.97 0.007 9.552 2 11.551 0.004

Ddit4 DNA-damage-inducible transcript 4 Rn01433735_g1 1.37 C 1 0.700 5.335 2 14.364 0.019

Dnaja1 Dnaj (Hsp40) homolog subfamily A Rn00576012_m1 1.3 Sh 0.90 0.001 5.781 2 13.511 0.015

Drd2 Dopamine receptor 2 Rn01418275_m1 0.42 0.98 0.144 3.866 2 10.660 0.055

Dusp1 Dual specificity phosphatase 1 Rn00678341_g1 0.51 C Sh 1.02 0.175 8.473 2 14.242 0.004

Egr1 Early growth response 1 Rn00561138_m1 0.57 C Sh 1.11 0.812 9.374 2 13.905 0.003

Eng Endoglin Rn01438763_m1 3.32 C Sh 1.02 0.025 19.435 2 13.210 0.0001

Epn2 Epsin 2 Rn00573059_m1 1.56 C Sh 0.95 0.512 10.339 2 14.306 0.002

Faah Fatty acid amide hydrolase Rn00577086_m1 0.91 0.97 0.759 0.173 2 14.362 0.842

Fgf2 Fibroblast growth factor 2 Rn00570809_m1 2.06 C Sh 0.94 0.523 29.098 2 14.232 0.0001

Fos FBJ osteosarcoma oncogene Rn02396759_m1 0.34 C Sh 1.09 0.001 27.985 2 11.022 0.0001

Gabbr1 Gamma-aminobutyric acid (GABA)

B receptor 1

Rn00578911_m1 1.11 0.98 0.768 0.708 2 14.522 0.509

Gad1 Glutamate decarboxylase 1 Rn00690300_m1 1.22 0.94 0.319 1.502 2 13.964 0.257

(Continued)

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rTMS Effects on Gene Expression in Stroke in Rats

Table 1. (Continued)

Gene
Symbol

Gene name Assay ID MCAO Group
Sig. Diff.

ShMCAO Variance Statistic

(F)

df1 df2 Sig.

Gad2 Glutamate decarboxylase 2 Rn00561244_m1 1.11 0.9 0.025 0.978 2 13.791 0.401

Gadd45b Growth arrest and DNA-damage
inducible

Gapdh Glyceraldehyde-3-phosphate

dehydrogenase

Rn01452530_g1 1.79 C 1.67 0.306 15.568 2 13.916 0.0001

Rn01775763_g1 0.92 0.89 0.010 0.125 2 13.561 0.884

Gfap Glial fibrillary acidic protein Rn00566603_m1 14.56 C Sh 0.95 0.002 51.164 2 12.655 0.0001

Gls Glutaminase Rn00561285_m1 1.46 Sh 0.85 0.038 5.336 2 13.834 0.019

Gnmt Glycine N-methyltransferase Rn00567215_m1 2.07 C Sh 0.77 0.700 7.724 2 14.462 0.005

Gpx1 Glutathione peroxidase 1 Rn00577994_g1 4.34 C Sh 0.92 0.002 15.431 2 12.630 0.0001

Gpx2 Glutathione peroxidase 2 Rn00822100_gH 6.14 C Sh 0.87 0.007 8.303 2 13.333 0.005

Gria1 Glutamate receptor ionotropic

AMPA 1

Gria2 Glutamate receptor ionotropic

AMPA 2

Gria3 Glutamate receptor ionotrophic

AMPA 3

Gria4 Glutamate receptor ionotrophic

AMPA 4

Grin2a Glutamate receptor ionotropic

NMDA2A

Grin2c Glutamate receptor ionotropic N
methyl D-aspartate 2C

Hif1a Hypoxia-inducible factor 1 alpha

subunit

Rn00709588_m1 1.07 0.94 0.305 0.401 2 13.553 0.677

Rn00568514_m1 0.82 0.9 0.022 0.468 2 12.949 0.636

Rn00583547_m1 1.15 0.85 0.006 1.487 2 13.644 0.261

Rn00568544_m1 1.05 0.86 0.073 0.406 2 13.482 0.674

Rn01424654_m1 0.73 0.95 0.110 3.112 2 13.641 0.077

Rn00561359_m1 1.29 Sh 0.93 0.029 4.460 2 13.883 0.032

Rn00577560_m1 1.75 C Sh 0.87 0.012 21.840 2 13.125 0.0001

Hmox1 Heme oxygenase 1 Rn00561387_m1 17.62 C Sh 0.97 0.004 97.867 2 13.148 0.0001

Hspa4 Heat shock protein 4 Rn00596544_m1 1.79 C Sh 0.97 0.124 11.270 2 14.233 0.001

Hspd1 Heat shock protein 1 Rn00821037_g1 1.47 Sh 0.87 0.246 3.891 2 13.776 0.046

Hsph1 Heat shock 105/110 protein 1 Rn01412930_m1 1.03 0.89 0.011 0.561 2 13.668 0.583

Irf1 Interferon regulatory factor 1 Rn00561424_m1 12.89 C Sh 0.93 0.025 53.003 2 11.807 0.0001

Jun Jun oncogene Rn00572991_s1 0.25 C Sh 0.98 0.060 58.108 2 9.951 0.0001

Junb Jun B proto-oncogene Rn01251660_s1 5.44 C Sh 1.08 0.021 53.484 2 13.243 0.0001

LOC246295 Glycine- glutamatethienylcyclohexylpiperidine-binding

protein

Rn00595357_m1 1.38 Sh 0.93 0.060 5.062 2 14.004 0.022

Mapk1 Mitogen activated protein kinase 1 Rn00671828_m1 1.17 0.9 0.001 2.375 2 12.216 0.135

Mgll Monoglyceride lipase Rn00593297_m1 1.30 1 0.852 2.540 2 14.556 0.113

Mmp19 Matrix metallopeptidase 19 Rn01756324_m1 34.16 C Sh 0.9 0.098 515.110 2 11.774 0.0001

Mmp2 Matrix metallopeptidase 2 Rn01538170_m1 14.69 C Sh 0.89 0.005 23.448 2 12.486 0.0001

Mmp3 Matrix metallopeptidase 3 Rn00591740_m1 2.63 C Sh 0.92 0.765 15.743 2 14.195 0.0001

Mmp9 Matrix metallopeptidase 9 Rn00579162_m1 0.98 0.87 0.082 0.207 2 13.822 0.815

Myc Myelocytomatosis oncogene Rn00561507_m1 7.30 C Sh 0.93 0.016 36.603 2 11.794 0.0001

Napepld N-acyl phosphatidylethanolamine

phospholipase D

Rn01786262_m1 1.54 Sh 0.86 0.096 4.406 2 14.123 0.033

Nog Noggin Rn01467399_s1 1.09 0.89 0.006 1.460 2 13.229 0.267

Nos1 Nitric oxide synthase 1 neuronal Rn00583793_m1 1.19 0.98 0.759 1.378 2 14.584 0.283

Nptx2 Neuronal pentraxin 2 Rn01756377_m1 0.63 C 1.03 0.213 5.060 2 13.938 0.022

Per3 Period homolog 3 (Drosophila) Rn00709499_m1 1.91 C Sh 0.88 0.032 8.803 2 13.696 0.003

Plat Plasminogen activator tissue Rn00565767_m1 2.00 C Sh 1.03 0.130 9.461 2 13.679 0.003

Pxmp4 Peroxisomal membrane protein 4 Rn00597183_m1 1.39 C Sh 0.97 0.771 5.119 2 14.557 0.021

(Continued)

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 8 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Table 1. (Continued)

Gene
Symbol

Gene name Assay ID MCAO Group
Sig. Diff.

ShMCAO Variance Statistic

(F)

df1 df2 Sig.

Rdx Radixin Rn01766742_m1 1.78 Sh 0.84 0.001 11.055 2 12.924 0.002

Slc3a1 Solute carrier family 3 member 1 Rn00568087_m1 3.82 C Sh 1.06 0.003 7.509 2 12.070 0.008

Tacr1 Tachykinin receptor 1 Rn00562004_m1 0.95 0.98 0.808 0.038 2 14.501 0.963

Tgfa Transforming growth factor alpha Rn00446234_m1 1.65 C Sh 0.98 0.929 10.059 2 14.658 0.002

Tnf Tumor necrosis factor Rn99999017_m1 5.71 C Sh 0.86 0.001 14.610 2 12.499 0.001

Trpv1 Transient receptor potential cation

channel subfamily V

Rn00583117_m1 1.42 0.92 0.116 1.039 2 12.134 0.383

Tubb5 Tubulin beta 5 Rn00597407_m1 0.96 0.93 0.061 0.069 2 13.227 0.933

Vegfa Vascular endothelial growth factor

A

doi:10.1371/journal.pone.0139892.t001

Rn00582935_m1 1.01 0.91 0.009 0.293 2 13.496 0.751

scale (BS) at 48h, between groups with and without MCAO were evaluated using Kruskal-Wallis H test. Changes in neurological deficits assessed by BDS score as categorical variable were
analyzed in two nested trials using regression (analysis of covariance). The first trial sought to
study the effects of MCAO on BDS before stimulation at 48h. For this BDS scores were
regressed on MCAO, and their corresponding baseline values at -24h. The second trial aimed
to examine the effects of rTMS on BDS after stimulation, i.e. at 17d. For this BDS at 17d were
regressed on the categorical variable stimulus and their corresponding “baseline” value at 48h
(only for groups with MCAO). Post-hoc comparison was done using Tukey's method in order
to adjust for multiple comparisons.

We separated the statistical analysis of changes in gene expression (RQ values) into two parts,
one comparing changes between the control, stroke and sham-stroke groups and one comparing
changes between the stroke, rTMS and sham-stimulated groups. All changes in RQ values for all
groups were initially examined for outliers; if present, they were excluded. We applied a one-way
Welch ANOVA (robust test of equality of means) because in all but two of the examined genes,
the assumption of homogeneity of variances was violated (Levene's Test of Homogeneity of
Variance p<0.05) and because the RQ values for the majority of genes were not normally distributed (Shapiro-Wilk test of normality p<0.05). To rigorously control for multiple comparisons,
the Welch ANOVA was followed by a Games-Howell post-hoc test for multiple comparisons.
Changes were considered significant if p<0.05. In order to reduce the number of groups for comparison, the gene expression data and the BS and BDS data on functional deficits from all shamstimulated groups were combined into a single sham-stimulated group.

Results

Physiological data

All results are given as the mean ± SEM. There were no significant differences (mixed factor
ANOVA p<0.05) between the ShMCAO, MCAO, ShSTIM and rTMS groups during surgery (rectal temperature of ShMCAO = 36.8±0.1°C, MCAO = 36.7±0.15°C, ShSTIM = 36.9±0.15°C, and all
rTMS groups = 36.8±0.15°C) or during the recovery period (rectal temperature of ShMCAO = 36.6
±0.15°C, MCAO = 36.5±0.15°C, ShSTIM = 36.6±0.15°C, and all rTMS groups = 36.6±0.1°C).

Assessment of the stroke and the effect of iTBS on stroke volume

On average the stroke involved 41.2% (range 40.3–44.6%, n = 6) of the left hemisphere total
volume at 48h. TTC staining revealed significant brain damage that included the entire MCA

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 9 / 25

rTMS Effects on Gene Expression in Stroke in Rats

territory in the parietal cortex. The injured areas typically involved cortices (frontal, parietal,
temporal and parts of the occipital), striatum and parts of the thalamus and hypothalamus of
the left hemisphere (Fig 2A). There was no visible lesion in sham-operated animals (n = 3).

Immunostaining was performed to further verify the MCAO and was in accordance with
previously reported results. In brief, immunostaining of sections of the contralateral non-ischemic hemisphere showed no Tnf immunoreactivity whereas Iba1- positive cells showed ramified
morphology of non-activated microglia and astrocytes with Gfap positive processes. In comparison, on the lesioned side the Tnf immunoreactivity was remarkably up-regulated with activated astrocytes and microglia in the peri-infarct zone (Fig 2B). The triple
immunofluorescence labeling showed that Tnf immunoreactive processes were also Gfap positive (marker of astrocytes) but none of them were Iba1 positive (marker of microglia) confirming that Tnf was up-regulated in astrocytes and not in microglia.

Finally, to assess the effects of rTMS on the size of the infarcted area, in a separate group of
animals (n = 14), brains were TTC stained after two weeks of iTBS (17d) and compared to
MCAO only group. On average, at 17d the infarcted area involved 43.8% (range 41.3–46.6%,
n = 7) of the left hemisphere’s total volume in MCAO animals. iTBS applied for two weeks had

Fig 2. Photographs of TTC staining (A) showing sham-operated (upper panel) and MCAO brain (lower panel), and immune-staining (B) of Gfap and
Tnf in the cerebral cortex from MCAO animal. The inset in the lower panel (A) indicates the typical region of the cortical tissue sampled for gene analysis
corresponding also to Gfap and Tnf immune-staining.

doi:10.1371/journal.pone.0139892.g002

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 10 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Fig 3. Changes in the behavior deficit score (BDS), on a 0-21point neurologic deficit scale where 0 indicates fully expressed deficit and 21
corresponds to normal animal, in MCAO, ShMCAO, 1 Hz, 5 Hz, cTBS, iTBS and ShSTIM groups prior to MCAO (-24h), 48 hours after MCAO (48h)
and two-weeks later (17d). *p<0.0005 compared to MCAO group.

doi:10.1371/journal.pone.0139892.g003

no significant effect on infarct size (p>0.05). The average infarct size in the iTBS group was
40.9% (range 39.4–44.1%, n = 7).

The effect of MCAO and rTMS on behavior

In terms of the 5-point neurological scale (BS) there was a significant change in median score
in MCAO groups (1Hz, 5Hz, cTBS, iTBS, ShSTIM) compared to groups without MCAO
(CON and ShMCAO) at 48h (p<0.0005 Kruskal-Wallis H test). The median BS was 3 in
MCAO group, while in non-MCAO group it was 0. Similarly, the dependence of BDS on baseline values at 48h was highly significant (p<0.0005). MCAO reduced the median 21 point neurologic deficit score (BDS) by 6.8 points at 48 hours (Fig 3). The second trial analysis of BDS at
17 days, showed highly significant effect of rTMS (F(1,19.153) = 2201.023, p<0.0005). cTBS and
iTBS performed significantly better, improving BDS in stimulated groups, compared to
MCAO, by 2.45 (p<0.0005) and 4.5 (p<0.0005) points, respectively.

Changes in gene expression following MCAO

The initial analysis compared changes between the stroke and sham-operated groups using the
control group as a calibrator. Out of the 96 genes, nine were not amplified and were excluded
from the analysis. MCAO induced significant change in the expression of 54 genes, out of
which 10 were significantly downregulated, and 44 significantly upregulated (Fig 4A).

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 11 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Fig 4. Fold-changes in mRNAs (RQs) of all genes that showed significant changes in expression after MCAO using the control group (CON) as a
calibrator (A) and of all genes that showed significant changes in expression after two weeks of rTMS using the MCAO group as a calibrator (B).

doi:10.1371/journal.pone.0139892.g004

We conducted a one-way ANOVA to determine if MCAO induced changes in expression of
genes. We initially assessed the data for outliers by inspecting the boxplot, and we assessed the
normality through a Shapiro-Wilk test. For all genes, in all three groups, the expression was
normally distributed, except for the following genes: Atf3, Btg2, Gadd45b, Irf1 in the ShMCAO
group, as well as Drd2 in both control and ShMCAO group (Shapiro-Wilk test, p<0.05).

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 12 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Table 1 illustrates the homogeneity of variances for each gene, as assessed by Levene's test of
homogeneity of variance. Considering that the assumption of homogeneity of variances was
violated for several genes, we conducted a one-way Welch ANOVA to determine if gene
expression differed between the groups. Group differences were examined by Games-Howell
post-hoc test for multiple comparisons (see Table 1, group difference column). In this study,
changes in gene expression were comparable to those reported earlier [41–43]. Highly-regulated genes were those related to inflammation, response to injury, brain ischemia, and regulation of angiogenesis, while no significant changes were found among genes related to
neurotransmission and plasticity. The highly upregulated genes included Adm (15-fold),
Angpt2 (26-fold), Atf3 (24-fold), Cxcr4 (8-fold), Gfap (15-fold), Gpx2 (6-fold), Hmox1
(17-fold), Irf1 (13-fold), Junb (5-fold), Mmp19 (34-fold), Mmp2 (15-fold), Myc (7-fold) and
Tnf (6-fold). The highly downregulated genes included Bdnf (2.5-fold), Fos (3.3-fold) and Jun
(5-fold).

Changes in gene expression following rTMS

We conducted a one-way ANOVA to determine if rTMS induced changes in gene expression
after MCAO and if they depend on the rTMS protocol. Similar to the previous analyses, we initially assessed the data for outliers by inspecting the boxplots and assessed normality with the
Shapiro-Wilk test. Only the expression of 8 genes was not normally distributed in one of the
groups (Shapiro-Wilk test, p<0.05). The expression of all other genes had normal distributions
in all groups. Table 2 shows the homogeneity of variances for each gene (Levene's test of homogeneity of variance). Because the assumption of homogeneity of variances was violated for
majority gene expression, a one-way Welch ANOVA was conducted to determine if the expression of genes differed between the groups. This test was followed by a Games-Howell post-hoc
test for multiple comparisons (see Table 2, group difference column).

Table 2 shows the results of the F statistics. rTMS induced significant upregulation in
expression of 52 genes (see also Fig 4B), while none showed significant downregulation. The
majority of changes in gene expression were found after iTBS. Here, only the major changes
will be summarized in textual form. Amongst the genes involved in angiogenesis, whose
expression was upregulated by MCAO alone, only Bai1, a p53-dependent angiogenesis inhibitor, showed further significantly increased expression after iTBS, while other genes were not
significantly affected by rTMS, including Angpt1 and Angpt2, Atf3, Cxcl12, Cxcr4, Eng and
Fgf2. Interestingly, Vegfa, an angiogenesis activator, was not altered by MCAO but significantly
upregulated by iTBS. With the exception of Tnf, none of the genes related to inflammation and
injury showed further significant changes following rTMS though they were upregulated by
MCAO (Bag3, CryaB, Gpx1, Gpx2, Hmox1, and Irf-1).

Several genes involved in neuroprotection, repair and tissue remodeling showed increased
expression after MCAO and were further upregulated by rTMS. Expression of Creb1, Epn2
and Junb increased after iTBS, and Plat increased after cTBS. Genes that showed no change in
expression after MCAO but were upregulated after TBS included Mapk1, involved in proliferation and differentiation, and Tubb5, which codes structural cytoskeleton microtubular proteins. Amongst the genes with expression significantly downregulated by MCAO, Bdnf was
significantly increased by 5Hz, cTBS and iTBS (a 6-fold increase), while Bai1, Fos and Jun were
upregulated only after iTBS.

The majority of genes showing increased expression following rTMS are involved in neurotransmission and plasticity across several neurotransmitter pathways. Among the examined
genes related to glutamate pathways, iTBS induced significant upregulation of Gls, which codes
for the enzyme directly involved in the presynaptic terminal conversion of glutamine to

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 13 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Table 2. Fold-changes in mRNAs (RQs) of all genes that showed expression on the microarray after two weeks of rTMS using the MCAO group as
a calibrator. MCAO, 1, 5, and Sh in the group significance column indicate significant difference to MCAO, 1Hz, 5 Hz, and ShSTIM groups, respectively.

Gene Symbol 1 Hz 5 Hz cTBS cTBS Gr. Sig. Dif. iTBS iTBS Gr.Sig. Diff. Variance F value df1 df2 Sig.

Actb 0.81 1.09 1.89 1.98 0.001 2.608 5 18.627 .059

Adcy1 0.72 1.11 1.43 2.28 0.001 2.048 5 18.497 .119

Adcy8 1.15 1.12 1.81 2.87 MCAO Sh 0.001 2.902 5 18.204 .043

Adm 1.35 0.83 2.05 1.49 0.011 1.050 5 18.624 .418

Adrb1 0.94 1.30 1.71 2.49 0.021 1.809 5 18.047 .162

Ak1 0.80 0.93 1.41 2.72 0.006 2.331 5 17.516 .086

Angpt1 1.00 0.81 1.98 2.19 0.001 1.237 5 17.871 .333

Angpt2 1.22 1.02 1.98 1.52 0.001 .595 5 18.244 .704

Apoe 0.78 0.80 1.09 1.63 0.001 2.214 5 18.715 .096

App 0.68 0.98 1.70 2.60 MCAO 1 5 Sh 0.001 4.777 5 17.729 .006

Arrb1 0.75 0.94 1.56 2.97 MCAO 1 Sh 0.001 3.921 5 17.865 .014

Arrb2 0.80 1.03 1.51 1.98 0.043 2.260 5 17.689 .093

Atf3 0.83 0.72 1.59 1.38 0.001 .958 5 18.072 .469

Bag3 1.03 0.90 1.96 2.25 0.001 1.905 5 18.631 .142

Bai1 0.60 0.91 1.71 2.42 MCAO 1 5 Sh 0.001 4.326 5 17.644 .010

Bdnf 1.43 2.62 3.17 5.82 MCAO 1 5 Sh 0.001 5.490 5 18.274 .003

Btg2 0.76 0.71 1.12 1.08 0.001 2.051 5 18.807 .117

Car11 0.77 0.97 1.09 2.41 0.001 1.896 5 18.153 .145

Cck 0.71 0.82 1.49 3.17 0.003 2.377 5 17.907 .080

Cdk5 0.67 0.78 1.58 2.80 MCAO 1 5 Sh 0.001 4.499 5 17.762 .008

Cited2 0.92 1.16 1.60 2.46 MCAO 1 5 Sh 0.001 3.003 5 17.933 .038

Clock 0.99 1.05 1.90 3.13 0.001 1.905 5 18.091 .143

Cnr1 0.84 0.82 1.63 3.03 1 5 Sh 0.001 3.471 5 18.098 .023

Creb1 0.86 1.05 2.20 3.25 MCAO 1 5 Sh 0.001 4.395 5 18.228 .008

Cryab 1.10 1.16 2.15 3.05 0.001 2.539 5 18.311 .065

Csnk1e 0.66 0.94 1.79 2.57 MCAO 1 5 Sh 0.001 7.680 5 17.957 .001

Cxcl12 1.15 0.85 1.68 1.75 0.006 1.046 5 18.772 .420

Cxcr4 0.74 0.88 2.04 1.57 0.001 1.440 5 18.543 .257

Dagla 0.63 0.88 1.93 2.94 MCAO 1 5 Sh 0.001 4.318 5 17.966 .009

Dbh 1.03 1.16 1.78 2.17 MCAO Sh 0.022 3.260 5 18.871 .027

Ddit4 1.18 0.80 1.29 1.62 0.003 1.707 5 18.063 .184

Dnaja1 1.06 1.04 1.81 3.66 MCAO 1 5 Sh 0.001 2.767 5 18.302 .050

Drd2 0.83 1.00 3.23 6.78 MCAO 1 5 Sh 0.001 3.328 5 18.093 .026

Dusp1 0.62 0.84 1.43 2.76 MCAO 1 5 Sh 0.001 5.989 5 18.358 .002

Egr1 0.82 1.20 2.10 3.17 MCAO 1 Sh 0.001 3.807 5 18.772 .015

Eng 0.70 1.10 1.93 1.29 0.003 1.555 5 18.305 .222

Epn2 1.21 1.12 1.82 3.38 MCAO Sh 0.001 3.045 5 18.090 .036

Faah 0.92 0.95 2.23 4.07 MCAO 1 5 Sh 0.004 4.819 5 18.256 .006

Fgf2 0.93 1.03 1.63 2.34 0.005 1.988 5 18.331 .129

Fos 1.73 1.73 3.78 MCAO 1 5 Sh 3.83 MCAO 1 5 Sh 0.001 11.754 5 18.411 .001

Gabbr1 0.68 0.88 2.34 1 5 3.37 MCAO 1 5 Sh 0.001 8.054 5 17.953 .001

Gad1 0.65 0.83 1.26 2.55 MCAO 1 5 0.001 2.871 5 17.762 .045

Gad2 0.69 1.02 1.84 2.92 MCAO 1 5 Sh 0.001 3.340 5 18.844 .025

Gadd45b 1.08 0.90 1.68 2.16 MCAO Sh 0.001 2.902 5 18.693 .042

Gapdh 0.88 1.00 1.63 3.13 0.001 2.135 5 18.251 .107

Gfap 0.92 0.91 1.81 1.87 0.001 1.014 5 18.364 .438

(Continued)

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 14 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Table 2. (Continued)

Gene Symbol 1 Hz 5 Hz cTBS cTBS Gr. Sig. Dif. iTBS iTBS Gr.Sig. Diff. Variance F value df1 df2 Sig.

Gls 0.58 0.85 1.34 3.35 MCAO 1 5 Sh 0.002 3.552 5 18.310 .020

Gnmt 0.64 0.77 1.11 3.45 MCAO 1 5 C Sh 0.001 4.819 5 17.912 .006

Gpx1 0.75 0.93 1.90 1.76 0.017 1.855 5 18.496 .151

Gpx2 1.12 1.30 4.25 2.44 0.002 .756 5 18.168 .593

Gria1 0.84 0.98 1.99 3.06 MCAO 1 5 Sh 0.001 4.960 5 18.035 .005

Gria2 0.86 1.20 2.12 3.16 MCAO 1 5 Sh 0.001 5.981 5 18.183 .002

Gria3 0.96 0.92 1.47 2.51 MCAO 1 5 Sh 0.002 3.095 5 18.325 .034

Gria4 0.97 1.03 1.55 3.19 MCAO 1 5 Sh 0.008 3.273 5 18.670 .027

Grin2a 0.65 0.95 1.87 2.91 MCAO 1 5 Sh 0.001 3.496 5 18.141 .022

Grin2c 1.18 0.99 1.80 MCAO 5 Sh 3.27 MCAO 1 5 Sh 0.001 5.490 5 18.153 .003

Hif1a 0.99 1.00 1.51 2.76 0.001 1.634 5 18.180 .201

Hmox1 0.53 0.76 2.98 0.88 0.002 2.247 5 17.712 .095

Hspa4 0.65 0.83 2.13 1 5 2.69 MCAO 1 5 Sh 0.001 7.314 5 18.265 .001

Hspd1 0.76 0.86 1.65 1.92 MCAO 1 5 Sh 0.001 3.841 5 18.581 .015

Hsph1 0.72 0.97 1.48 2.68 MCAO 1 5 Sh 0.001 3.491 5 18.539 .021

Irf1 0.78 0.77 1.96 1.59 0.001 1.337 5 18.059 .294

Jun 0.91 0.91 1.40 2.16 MCAO 1 5 Sh 0.002 2.611 5 18.717 .050

Junb 0.79 0.86 1.65 1.62 1 5 0.001 3.717 5 16.954 .019

LOC246295 0.88 0.96 2.36 3.47 MCAO 1 5 Sh 0.010 6.647 5 18.162 .001

Mapk1 0.56 1.10 3.74 4.51 MCAO 1 0.001 4.210 5 17.927 .010

Mgll 0.70 0.96 1.33 2.61 MCAO 1 5 Sh 0.004 3.509 5 18.487 .021

Mmp19 0.65 0.84 1.86 5 1.35 0.001 2.797 5 17.948 .049

Mmp2 0.93 1.01 1.97 1.32 0.001 .846 5 18.736 .534

Mmp3 0.64 1.01 1.09 1.58 0.009 1.589 5 18.606 .212

Mmp9 0.77 1.02 1.77 2.81 MCAO Sh 0.001 4.087 5 18.531 .011

Myc 1.02 1.13 1.35 1.84 0.009 .612 5 18.445 .692

Napepld 0.75 1.19 1.59 2.77 MCAO 1 5 Sh 0.005 3.529 5 17.677 .022

Nog 0.65 0.85 1.76 2.05 MCAO 1 5 0.001 3.928 5 17.920 .014

Nos1 0.86 0.84 2.28 3.05 MCAO 1 5 Sh 0.001 3.852 5 18.218 .015

Nptx2 0.77 1.11 1.88 4.14 0.002 2.142 5 18.198 .106

Per3 0.89 0.87 1.42 2.37 MCAO 1 5 0.006 3.046 5 17.845 .037

Plat 0.67 0.80 1.97 1 1.36 0.001 4.281 5 18.757 .009

Pxmp4 0.77 0.89 1.67 2.70 MCAO 1 5 Sh 0.002 3.997 5 18.550 .012

Rdx 1.06 1.08 1.83 3.03 0.001 2.255 5 18.160 .093

Slc3a1 1.31 1.44 3.37 4.40 MCAO 1 5 Sh 0.001 3.319 5 18.242 .026

Tacr1 0.87 0.85 2.43 3.22 0.001 2.406 5 18.727 .076

Tgfa 0.97 0.96 2.17 3.33 0.001 2.599 5 18.655 .060

Tnf 0.29 S,C,I 0.68 2.27 2.06 MCAO 1 5 Sh 0.001 8.515 5 17.045 .001

Trpv1 1.52 0.97 2.11 5 3.87 MCAO 1 5 Sh 0.001 6.265 5 18.186 .002

Tubb5 0.76 1.04 1.68 2.43 MCAO 1 5 Sh 0.001 4.506 5 17.862 .008

Vegfa 1.15 1.17 1.94 3.28 MCAO 1 5 Sh 0.001 7.427 5 18.018 .001

doi:10.1371/journal.pone.0139892.t002

glutamate. The mRNAs of all four subunits (Gria 1–4) of the glutamate ionotropic AMPA
receptor, as well as mRNAs of the glutamate NMDA receptor (Grin2a and 2c), were upregulated after iTBS. Upregulated mRNAs related to GABA signaling included Gabbr1, which
codes the component of the heterodimeric G-protein coupled GABA receptor, and Gad1 and

PLOS ONE | DOI:10 1371/journal pone 0139892 October 2 2015 15 / 25

rTMS Effects on Gene Expression in Stroke in Rats

Gad2, which encode the glutamic acid decarboxylase responsible for catalyzing GABA biosynthesis. Several genes involved in the endocannabinoid system were also upregulated. The following were all significantly upregulated after iTBS: Dagla, which encodes a diacylglycerol
lipase enzyme involved in the generation of anandamide; Faah, which catalyzes degradation of
the endocannabinoid class of signaling lipids; and Cnr1, a type 1G protein-coupled cannabinoid receptor activated by the endocannabinoid neurotransmitters like anandamide, and
Napepld involved in endocannabinoid biosynthesis. Upregulated genes related to neuronal
plasticity and GPCR signaling included the following: App, involved in regulation of synapse
formation and plasticity though regulation of Ca 2+ channels; Arrb1, involved in agonist-mediated desensitization and downregulation of GPCR receptors; and Drd2, a dopamine receptor
D2. Finally, after iTBS, the neurotrophin Bdnf and other genes related to Bdnf pathways were
upregulated, including Adcy8 involved in the modulation of G protein activity and Gadd45b
involved in the regulation of growth.

QRT-PCR gene expression

Changes in expression, comparable to those detected by the low-density arrays, were confirmed
in three genes examined by fast real-time RT-PCR. Increases in expression were seen in
Gabbr1 (3.29-fold), Gria2a (4.13-fold) and Bdnf (6.26-fold), respectively. These findings were
similar to the fold-changes seen with the low-density array of 3.37, 3.16 and 5.82-fold for these
three genes, respectively. The degree of RQ variation between repeated samples from different
animals was below 0.5.

Discussion

The principal findings of this study have demonstrated that rTMS has the potential to influence
the expression of genes involved in regulating multiple and diverse pathways and processes,
including those involved in the brain response to ischemia. They also show that changes
depend on rTMS pattern and frequency with only iTBS inducing significant changes in gene
expression. The results confirm and extend earlier suggestions that rTMS in stroke may
enhance the overall brain response to ischemic injury. They also suggest that this may be facilitated through activation of diverse neuronal and network level pathways spanning neuroprotection, cellular repair, remodeling and neuronal plasticity.

MCAO induced lesion and the neurological deficits

The transient MCAO is a widely-used model of stroke because it closely mimics human stroke
induced by occlusion of a large artery [44,45]. In the present study, MCAO induced reproducible cortical injury similar in extent and localization to reported data [45–47]. Namely, the
pattern of TTC staining and the volume of infarcted brain tissue did not differ significantly
between animals 48 hours after MCAO. It should be noted though that in this study TTC staining was performed 48 hours after MCAO. This may have affected the detection of the infarcted
area and the volumetry as it was suggested that TTC should not be used beyond 24 hours since
the inflammatory cells harbor intact mitochondria [48,49]. Damage to such widespread and
functionally diverse brain regions produced significant motor impairment, as evidenced by significant increase in 5-point neurological deficit score (median BS = 3) and significant decrease
(by 6.8 points) of 21-point BDS score at 48h in all MCAO groups. The character and the extent
of neurological deficits were comparable to previously reported ones [31–33,35].

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rTMS Effects on Gene Expression in Stroke in Rats

Changes in gene expression after MCAO

The number of genes reported in studies examining global gene expression changes after cerebral ischemia varies considerably spanning 150 [50] to 700 genes [42]. Furthermore, regulated
genes show large variations in expression depending on postischemic time [41]. It was also
shown that gene expression in the rat brain, although similar, differs between cortical regions,
with 30 genes found to be enriched in the frontomedial cortex [51,52]. In this study, genes were
selected based on their distinct appearance in the frontal cortex [51,52] and level of modulation
by ischemia [41,43,53]. Selection was also based on the limited data on genes shown to be regulated by rTMS [38,54,55]. We also included several genes that may show regulation by rTMS,
including those involved in regulation of synaptic plasticity. Thus, although we examined
fewer genes than traditionally reported, these genes provided broad insight into the effects of
rTMS on the ischemic brain.

Stroke alone induced changes in gene expression spanning several functional categories,
which were comparable to changes in gene expression reported in earlier studies [41–43,56,57].
For example, several genes related to angiogenesis, a natural defense mechanism set to restore
oxygen and nutrient supply after stroke, were significantly upregulated two weeks after MCAO.
Other regulated genes included immediate early genes, heat shock proteins, anti-oxidative
enzymes, trophic factors, and genes involved in RNA metabolism, inflammation and cell signaling. The expression of Gfap, an established marker of astrocyte activation in brain ischemia, also
increased significantly, further suggesting that the developed model was reproducible and comparable to already-established changes in ischemia, providing suitable substrate to explore the
effects of rTMS.

rTMS induced changes in gene expression in stroke

Two weeks of rTMS induced significant increases in expression of a number of genes across
several functional categories. The effects of rTMS on genes related to angiogenesis may appear
quite limited, as judged by the significant upregulation of only Bai1 and Vegfa, yet they deserve
further attention. Namely, although none of the changes were significant, Angpt1 showed a
2-fold change after cTBS and a 2.2-fold change after iTBS, while Angpt2 reached a 2-fold
change after cTBS. Other genes in this functional group like Cxcr4 and Fgf2 were upregulated
by stroke and further increased after rTMS, but did not reach statistical significance. The fact
that the expression of these genes changed in TBS groups, but not in the 1Hz and 5Hz groups,
suggest that rTMS may affect angiogenesis-related genes. This finding is partly corroborated by
the significantly increased expression of Vegfa after iTBS, which is involved in regulation of
Angpt2.

The former findings also apply to genes involved in inflammation and injury. The genes
that were upregulated by stroke were not significantly changed by rTMS, except for Tnf, which
encodes a multifunctional proinflammatory cytokine known to be involved in the regulation of
a wide spectrum of biological processes including cell proliferation, differentiation and apoptosis. Interestingly, although not reaching significance, GFAP expression was 1.8-fold higher
after cTBS and iTBS, while there were no changes after 1Hz and 5Hz (0.9-fold change) (see
Table 2). Earlier studies showed that acute application of rTMS (25 Hz) induced a profound
but transient increase in GFAP [58], while chronic (11 weeks) rTMS had no effect [25]. Current results suggest the opposite effect, as if TBS may increase GFAP expression after two
weeks of stimulation in MCAO, while low-rate rTMS (1Hz and 5Hz stimulation) has no effect.

Among the genes related to neuroprotection, cellular repair and remodeling, rTMS significantly influenced expression of several genes, including the immediate early genes Fos, Jun
and JunB. In normal healthy animals, Fos expression was upregulated after single exposure to

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rTMS Effects on Gene Expression in Stroke in Rats

1 and 10 Hz rTMS [38], as well as after chronic (14 days) exposure of 20 Hz rTMS [54]. In
MCAO, chronic (7–28 days) exposure of 0.5 Hz rTMS induced significantly increased expression of Fos [59]. In the present study, Fos increased after TBS, but not after 1 and 5 Hz rTMS,
suggesting that the effects may depend on stimulation frequency and/or stimulation pattern.
Upregulation of Jun expression, which was also demonstrated earlier after acute rTMS [60],
suggests that it may interact with downstream apoptotic mechanisms [43] and potentially the
brain’s metabolic response to ischemia [61]. Research has also shown that rTMS may produce
increased neuronal survival and a smaller infarcted area after MCAO ischemia-reperfusion

[62]; it may also significantly increase expression of the antiapoptotic factor Bcl-2 in the
infarcted area, associated with improved learning and memory [63]. Other studies have also
suggested antiapoptotic effects of rTMS in subacute cerebral ischemia [24] and bilateral
MCAO in rats [23]. Extending these findings, this study reports changes in JunB and other
genes related to apoptosis, cellular repair and remodeling (Creb1, Epn2, Mapk1, Plat, and
Tubb5), suggesting that rTMS may have more profound effects on apoptosis and remodeling
in MCAO. Along the same lines is the finding of upregulation of MMP-9, which in chronic
stroke may be involved in neurovascular remodeling, thus promoting brain tissue repair and
regeneration during delayed phases after stroke [64]. Finally, GADD45, which is involved in
modulation of the cell response to stress was also upregulated by iTBS. Thus, it appears that
rTMS may support endogenous mechanisms of neurovascular remodeling in the perilesional
cortex as intrinsic (adaptive) repair responses become activated after injury. The main impact
of rTMS after stroke was found in the expression of genes related to neurotransmission and
plasticity, most of which were not changed by MCAO alone. In the glutamatergic pathway, the
effects of rTMS suggest increased presynaptic glutamate production (upregulation of Gls) and
NMDA (Gria 1–4) and AMPA (Grin2a and 2c) receptor expression. The ability of rTMS to
induce changes in brain activity that last after stimulation relates to changes in synaptic plasticity [65–68], similar to long-term potentiation and depression-like mechanisms [69,70]; thus, it
involves modification of activation and activity in the NMDA receptor systems [67,71].
Enhanced functional and structural plasticity after rTMS is also supported by data showing
that long-lasting rTMS induces increased glutamatergic synaptic strength in the hippocampus,
accompanied by structural remodeling of dendritic spines [72]. The results of changes in gene
expression in our study strongly suggest enhanced synaptic plasticity after rTMS in stroke.
This possibility is further corroborated by the significant functional recovery at 17d of animals
in the iTBS group. In the GABAergic pathway, rTMS significantly upregulated the GABAB
receptor (Gabbr1) mRNA, as well as genes involved in the control of GABA biosynthesis (glutamate decarboxylase, Gad1 and Gad2) in the cortical inhibitory interneurons of normal rats,
single application of rTMS affects the expression of glutamate decarboxylase isoforms GAD67
and GAD65, activity-dependent proteins which are coded by Gad1 and Gad2 [73]. Furthermore, changes in the expression of these two glutamate decarboxylase isoforms (GAD67 and
GAD65) follow complex patterns after rTMS [74], which also depend on the duration of rTMS

[75]. While the effects of increased expression of genes related to GABA may be related to its
general role in enhancing neuroplasticity and promoting functional recovery, its effects in
stroke may require optimal timing, as recently suggested [76].

The data regarding effects of rTMS on the endocannabinoid system are sparse and have not
been reported previously, particularly not in stroke. The main effects of rTMS seem to enhance
endocannabinoid biosynthesis (upregulation of Dagla, Napepld and Faah) as well as cannabinoid receptor activation and/or synthesis (upregulation of Cnr1). So far, the effects of rTMS on
the endocannabinoid system have been demonstrated in relation to depression, effects that
seem to be mediated through the Cb1 hippocampal cannabinoid receptor [77]. In stroke, the

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rTMS Effects on Gene Expression in Stroke in Rats

role of the endocannabinoid system is still poorly understood, but current evidence suggests
that its activation may be protective, particularly in relation to reperfusion injury [78].

Finally, the potential of rTMS in promoting neuroprotection and plasticity in stroke is further corroborated by the results that showed a significant increase in expression of genes
involved in GPCR signaling (Arrb1, Adcy8 and Bdnf). Bdnf is the most abundant neurotrophin
in the CNS and has been associated with numerous functions, such as synaptic plasticity, neurogenesis, motor neuron survival and plastic responses related to motor skill learning and hypoxic ischemic brain injury [79]. Bdnf is also involved in neuronal survival, protecting against
neuronal loss while increasing the function of surviving neurons [80]. iTBS induced significant
increases in expression of Bdnf (6-fold), whereas earlier studies showed increased Bdnf
expression after long-term rTMS in normal rats [81], and after 0.5 Hz rTMS after MCAO [59].
Altogether, our results suggest that rTMS increases expression of genes related to multiple processes that are likely to promote recovery after stroke.

Changes in gene expression and recovery of behavior deficits depend
on the characteristics of the rTMS protocol

Functional effects of rTMS on cortical excitability are determined by the stimulation intensity,
frequency and overall stimulation pattern. Studies in human subjects suggest that the direction
of changes depend on stimulation frequency and the theta-burst train pattern. High-frequency
(> 4 Hz) increases and low-frequency rTMS decreases the excitability [1], whereas an intermittent TBS train enhances, and a continuous TBS train tends to lower the cortical excitability [2].
One of the main aims of this study was to compare the effects of rTMS protocols, which
depress (5 Hz and iTBS) or potentiate (1Hz and cTBS) the cortical excitability. This assumes
that they may induce disparate effects on gene expression because they most likely differentially
affect NMDA-dependent synaptic plasticity. In each protocol, the number of pulses, overall
duration of the stimulation and the stimulation intensity were the same. Therefore, the results
demonstrate that changes in gene expression in stroke depend primarily on the stimulation
pattern (iTBS vs. cTBS) and frequency (1Hz and 5Hz vs. iTBS). Furthermore, they are not
related to the direction of the immediate change in cortical excitability. Earlier studies also suggested that long-term 1 Hz stimulation has limited effects on the cortical markers of neuroplasticity [82]. Meanwhile, increased GABA cortical signaling was also reported after long-term
1Hz rTMS, suggesting strengthened cortical inhibition in normal rats [73]. In addition to the
stimulation frequency, the absence of effects for chronic 1 HZ stimulation in this study may
also relate to altered cortical conditions due to stroke. Conspicuously, several genes showed
decreased expression (22 genes with <0.7 fold change) after 1 Hz stimulation, whereas no
genes had decreased expression after TBS. Although cTBS appears to be rather ineffective in
altering gene expression compared to iTBS, it should be noted that a considerable number of
genes were altered after cTBS without reaching significance. The potential difference in the
effectiveness between cTBS and iTBS may relate to their cell-type specific effects, particularly
in terms of cortical inhibitory interneurons [73], causing differential modulation of their activity [83] and differential activation of pathways in stroke. Differential effects of TBS patterns
may also depend on the synaptic connectivity and preferred discharge pattern for these inhibitory neurons [83]. In stroke, this physiological difference may have been further potentiated,
rendering certain cortical inhibitory subsystems more or less susceptible to stimulation.

The results of changes in neurologic deficits (BDS) after MCAO and two weeks of rTMS
further corroborate the aforementioned conclusions. The improvement in BDS were significantly superior after iTBS stimulation compared to other stimulated groups, strongly arguing
for its effectiveness in promoting recovery.

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rTMS Effects on Gene Expression in Stroke in Rats

Methodological aspects and limitations of this study

Brain tissue for gene expression analysis was taken from the cortical areas surrounding the
injured core. This method was considered appropriate because the characteristics of the cortical effects in MCAO with reperfusion in rats closely resemble the time course and clinical realities of human stroke. Namely, in MCAO with reperfusion, the ischemic core involves the
striatum, which remains densely ischemic, while the overlying cortex undergoes delayed, progressive neuronal death, practically representing ischemic penumbra in this model [84].
Changes in dorsolateral cortex are associated with delayed inflammatory mediators of ischemic
cell death and with induction of many hypoxia-induced genes involved in neuroprotection

[47]. These genes are induced solely in the cortex, making it the main target for neuroprotective
therapies. Because rTMS cannot selectively target the core, penumbra or healthy tissue surrounding the penumbra, it instead affects all of them. Therefore, it appeared warranted to sample changes in gene expression from both the penumbra and healthy surrounding tissue, as
they hold restorative potential after stroke.

The use of rTMS in stroke is based on the assumption that it may improve motor function by
facilitating the volitional recruitment of corticomotor output neurons, and/or that it may help to
re-establish a functional balance between the damaged and undamaged hemisphere. The second
assumption, based on the interhemispheric imbalance model [6,85] argues for the use of rTMS to
suppress the excitability of the contralesional hemisphere or to boost cortical excitability in the
ipsilesional hemisphere, releasing it from excessive interhemispheric inhibition. Although the
current study was not designed to explore this hypothesis, it showed the relative ineffectiveness
of cTBS in altering gene expression in the lesioned hemisphere. In human chronic stroke, cTBS
applied over the non-lesioned primary motor cortex had no effect on the cortical excitability of
the motor cortex of the affected hemisphere [86]. However, in acute stroke, cTBS induced a facilitation when applied over the cortical motor hand area of the intact hemisphere [87].

It should also be noted that no significant difference was found in the volume of MCAOaffected brain tissue after rTMS compared to MCAO alone at 17d; there was, however, a trend
toward reduction. Earlier studies that examined the spatio-temporal dimensions of ischemic
regions showed a similar distribution and size of lesioned areas 7 days after MCAO [47], thus
observed changes in volume may be more related to natural evolution of the ischemic injury
rather than to rTMS effects.

Certain limitations of the study should be considered when interpreting the results. Excitatory
and inhibitory rTMS effects on cortical excitability are primarily based on their immediate effect
on the normal brain when probed by MEP. At present, the extent to which they follow the same
pattern in a brain stroke remains unclear. Nevertheless, the current results provide insight into
the modulation of gene expression by these rTMS protocols, demonstrating that these changes
most likely do not depend on the direction of initial changes in excitability. rTMS was performed
by standard stimulation coil in this study, causing activation of not only the cortex but also of
subcortical structures due to the small size/volume of the rat brain [62,88]. To this ends, an ancillary observation relates to changes in expression of genes in the contralateral hemisphere after
rTMS (not shown), which showed no significant changes. Finally, having found changes in
expression of more than 50 genes, the study could not establish and quantify the extent to which
these genes lead to changes in related proteins; thus, further studies are needed to establish the
functional outcomes of changes in gene expression described in this study.

Conclusions

In summary, the results show that mapping of the gene response may provide significant
insight into alteration of molecular pathways and the effects of various rTMS protocols on

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rTMS Effects on Gene Expression in Stroke in Rats

these pathways in brain ischemia. The results also show the differential effectiveness of various
rTMS protocols in inducing changes in gene expression after ischemic-reperfusion brain
injury, emphasizing the importance of choice of rTMS protocol when applied over the lesioned
stroke hemisphere as therapeutic intervention. Furthermore, they show that the iTBS protocol
is the most effective one, affecting a multitude of genes, including those involved in angiogenesis, response to injury, cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. Further studies are needed to underpin the mechanisms of rTMS
modulation of gene expression and the eventual role of final products of gene activity in recovery after ischemic brain injury.

Author Contributions

Conceived and designed the experiments: MRL TEA. Performed the experiments: AJ JO KP SS
MRL. Analyzed the data: JO MRL SS NN. Contributed reagents/materials/analysis tools: MRL
SS. Wrote the paper: MRL TEA JO AJ SS NN.

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